Published February 1, 1995
Phosphatidic Acid Signaling Mediates Lung Cytokine
Expression and Lung Inflammatory
Injury After Hemorrhage in Mice
By Edward Abraham,* Stuart Bursten,$ Robert Shenkar,*
Janet Allbee,* Rubin Tuder, Paul Woodson,$ David M. Guidot,~
Glenn Rice,$ Jack W. Singer,g and John E. Repine*~
From the "Division of Pulmonary Sciences and Critical Care Medicine, *l~bb-Waring Institute
for Biomedical Research, University of Colorado Health Sciences Center, Denver, Colorado
80262; and SCell Therapeutics, Inc., Seattle, Washington, 98119
Summary
~
ute edematous lung injury (ARDS) 1 is a highly fatal
(mortality >50%) disorder, characterized by increased
lung cytokine levels and massive neutrophil accumulation in
the lung (1). Blood loss, such as occurs after severe injury,
even if resuscitated, is associated with increased expression
of proinflammatory cytokines, such as TNF-ol and IL-1/~, and
the frequent development of acute lung injury (2-5). Although
ARDS often occurs after an ischemia (hypoxia)-reperfusion
(reoxygenation) insult caused by hemorrhage and resuscitation, the relationship between hypoxia, cytokine activation,
neutrophil recruitment, and lung injury is unknown.
Certain species of PA that do not contain arachidonate in
either the sn-1 or sn-2 positions are produced from lyso-PA
by the membrane associated enzyme, lysophosphatidic acid
acyl transferase (LPAAT). Each system in which PA formation
via LPAAT has previously been demonstrated (i.e., IL-lfl, lipid
A or hydrophobic chemotherapeutic compounds) either involves activation of phospholipase A2, or causes membrane
perturbation or release of reactive oxygen intermediates which
indirectly activate phospholipase A2 (6, 7). These PA species are then rapidly converted to 1,2 diacylglycerol (DAG)
by phosphatidate phosphohydrolase (PAPH) (7-9). Both PA
1Abbreviationsusedin thispaper: AM, alveolarmacrophages; ARDS, acute
edematouslung injury; IPMC, intraparenchymalpulmonary mononuclear
cells; LPAAT,lysophosphatidicacid acyltransferase;lyso-PA,lysophosphatidic acid; PA, phosphatidic acid.
569
and DAG function as intracellular second messengers and can
be distinguished from the end-products of the phosphatidyl
inositol pathway by the absence of sn-2 arachidonate (7, 8).
CT1501R, 1-(5-R-hydroxyhexyl)-3,7-dimethyl xanthine, is
a functional inhibitor of LPAAT activity which reduces specific
PA generation (8). For example, CT1501R suppresses the
generation of PA induced by bacterial lipopolysaccharide with
an IC50 of 0.5-0.6/zM in the murine monocytic leukemia
cell line, P388, in vitro (8). Adding CT1501R to lipopolysaccharide-stimulated blood mononuclear cells inhibited TNF-o6
IL-1B and I1.-6 release, as well as IL-1/~ and TNF-c~ mRNA
accumulation, without affecting IL-8 expression (10). Similarly, inhibiting PA formation with CT-1501R protected mice
from endotoxin lethality (8). Because of the apparent role
of PA as an intracellular second messenger in neutrophil activation (11) and cytokine dependent responses, we investigated
the effects of inhibiting PA generation on neutrophil function in vitro as well as cytokine expression and the development of acute lung injury after hemorrhage and resuscitation in vivo.
Materials and Methods
Human Neutrophil Isolation. Human neutrophils (PMN) were
prepared from heparinized blood obtained from consenting, healthy
human volunteers. Neutrophils were isolated and highly purified
(>99%) by Percoll gradient and differential centrifugation and
resuspended in HBSS.
J. Exp. Med. 9 The Rockefeller University Press 0022-1007/95/02/0569/07 $2.00
Volume 181 February 1995 569-575
9
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Because phosphatidic acid (PA) pathway signaling may mediate many basic reactions involving
cytokine-dependent responses, we investigated the effects of CT1501R, a functional inhibitor
of the enzyme lysophosphatidic acid acyhransferase (LPAAT) which converts lysophosphatidic
acid (Lyso-PA) to PA. We found that CT1501R treatment not only prevented hypoxia-induced
PA increases and lyso-PA consumption in human neutrophils, but also prevented neutrophil
chemotaxis and adherence in vitro, and lung injury and lung neutrophil accumulation in mice
subjected to hemorrhage and resuscitation. In addition, CT1501R treatment prevented increases
in mRNA levels and protein production of a variety of proinflammatory cytokines in multiple
lung cell populations after blood loss and resuscitation. Our results indicate the fundamental
role of PA metabolism in the development of acute inflammatory lung injury after blood loss.
Published February 1, 1995
570
amplified cDNA, the PCR product was analyzed by agarose gel
electrophoresis. The number of PCR cycleswere selected for the
cytokineproduct from each cellularpopulation so that the ethidium
bromide stained amplifiedDNA products were between barely detectable and below saturation. The gel was then captured for computer integration using a UVP System (UVP Inc., San Gabriel,
CA). Densitometryresultswere normalizedto those for the cytokine
MIMIC.
ELISA Assayfor Cytokines. After centrifugation, supernatants
from BAL were stored at -70~ ELISA for TNF-o~ and IFN-y
content in BAL supernatants used paired monoclonal anti-mouse
TNF-c~ (MP6-XT3 and biotinylated MP6-XT22) and IFN-3, (R46A2 and biotinylatedXMG1.2) antibodies (PharMingen,San Diego,
CA), and alkaline phosphatase-conjugated strepavidin (Southern
Biotechnology,Birmingham,AL), with recombinantmouseTNF-ot
and IFN-~ as standards. The sensitivityof the ELISAwas 10 pg/ml.
HistologicAnalyses. At 3 d after hemorrhage-resuscitation,mice
were killed and the pulmonary circulation flushedby injecting PBS
into the right ventricle. Lungs were formalin injected through the
trachea, then removed en bloc with the heart and placed into formalin. Hematoxylin and eosin sections were prepared and examined blindly by a pulmonary pathologist (RT). Grading of the histopathologic changes (HE stain x 92 magnification) in the lungs
(neutrophil infiltration, interstitial edema, and intraalveolarhemorrhage) was performed on a 0 to 3 scale (with 0 being normal and
3 being the most severe abnormality).
StatisticaIAnalyses. Data are presentedas mean _+ SEM. Groups
were compared using one way analysis of variance and either the
Student-Newman-Keuls or the Tukey-KramerMultiple Comparison tests for differencesbetween groups. A p value of <0.05 was
considered to be statistically significant.
Results and Discussion
To test the inhibition of LPAAT by CT1501R, we exposed
CT1501R-treated human neutrophils to hypoxia and then reoxygenation in vitro. After exposure to hypoxia for 60 min
and then reoxygenation for 20 min, human neutrophils had
increased PA concentrations with a retention time (ILl) characteristic of linoleate~enriched PA species (Fig. 1). Hypoxia-reoxygenated neutrophils preincubated with 10-100 #M CT1501R had decreased PA (Fig. 2 a) and increased lyso-PA (Fig.
2 b) concentrations compared to untreated neutrophils.
CT1501R treatment also reduced stimulated human neutrophil adherence (Fig. 3 a) and chemotaxis (Fig. 3 b), but not
neutrophil superoxide anion generation (Hg. 3 c), in vitro.
Severe hemorrhage causes a systemic hypoxia and then
replacement of blood reoxygenates ischemic tissues (19). A
frequent consequence of these events is the development of
ARDS which appears to derive from an oxidative insult involving neutrophils and/or xanthine oxidase (1). Because our
findings indicated that CT1501R treatment not only inhibited
the generation of PA species during hypoxia-reoxygenation
but also neutrophil function in vitro, we suspected that PA
participates in inflammatory lung injury after hemorrhage-resuscitation. Mice treated 1 h after severe hemorrhage
with CT-1501R given along with the previously removed
blood during the resuscitation phase had decreased (p <0.05)
interstitial lung edema (Fig. 4 a) and intraalveolar hemorrhage (Fig. 4 b) compared to untreated mice. Moreover, after
Lung InflammatoryMechanismsin Shock
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HPLC Analysis of PA and Lyso-PA. Human neutrophils were
placed in hypoxia (95% N2 + 5% CO2) and then reoxygenated
(21% 02 + 5% CO2). After neutrophils were fixed in ice-cold
methanol, lipids were extracted and separated by HPLC with a
Waters #-Poracil| silicacolumn, using an anisocraticgradient (7-9).
Lipidsin the column effluentwere monitored at 206-224 nm. HPLC
fractions were also analyzed by thin layer chromatography, amine
content, acylcontent, and mass spectrometry to confirm peak identities. Fast-atom bombardment mass spectrometry spectra were acquired using a VG 70 SEQ tandem hybrid instrument of EBqQ
geometry (VG Analytical, Altrincham, UK) as previouslydescribed
(7, 8). Mass of total PA varied from 0.65-0.75% in unstimulated
neutrophils, to 2.7-3.6% of total detectable lipids in stimulated
neutrophils (increasedby 3.5-5-fold); lyso-PAmass in neutrophils
pretreated with CT1501R and subjected to hypoxia-reoxygenation
could range as high as 7-8% of total detectable lipids.
NeutrophilFunctionStudies. Neutrophil chemotacticactivity was
determined by Boydenchamber assayand quantitated as the number
of migrating PMN/5HPF using zymosan-activated serum as the
chemoattractant (12). Neutrophil adherence was assessedby quantitating the percentage of neutrophils adhering to nylon fibers after
addition of phorbol myristate acetate (PMA 10-6 M; Sigma Chem.
Co., St. Louis, MO) (13). Neutrophil superoxide production was
determined by quantitating superoxidedismutaseinhibitablereduction of cytochrome c in response to PMA (10-6 M) (14).
Hemorrhage and ResuscitationEvaluation. After methoxyflurane
anesthesia, male BALB/c mice (Harlan-Sprague-Dawley, Indianapolis, IN) 8-14 wk old, had 30% of their calculatedblood volume
(,o0.55 ml for a 20-gm mouse) withdrawn via cardiac puncture
over 60 s (2, 15). Blood was collected in a heparinized (5 U) sytinge, kept at 37~ for 1 h, and then reinfused into the retroorbital plexus of the briefly (<1 min) reanesthetized mouse. Resuscitated survivors did not develop hemothorax, bleeding into the
pericardial space, lung or cardiac contusion. Control mice were
subjectedsimilarlyto anesthesiaand cardiacpuncture without blood
withdrawal, and then injected retroorbitally with heparin (5 U)
in 0.2 ml PBS.
SemiquantitativePCR Analysis of Cytokine mRNA Levels. Relative cytokine mRNA levelswere determined by semiquantitative
PCR on alveolar macrophages (AM), peripheral blood monocytes
(PBMC), and intraparenchymal pulmonary mononuclear cells
(IPMC) collected from mice 3 d after hemorrhage-resuscitation
(2, 4). AM were isolated by centrifugation of bronchoalveolar la,cage (BAL) obtained by injecting and then aspirating 1.0 ml PBS
intratracheaUy. PBMC were isolatedfrom heparinizedblood diluted
2:1 with PBS, pH 7.3, and layeredonto a lympholyte-m gradient.
After centrifugation at 600 g for 20 min at 15~ cells at the interface were collected, washed in RPMI 1640, counted, and assessed
for viability which was consistently greater than 98% by trypan
blue exclusion. IPMC were isolated by collagenase digestion and
Percoll gradient purification (16). mRNA was extracted from isolated cells using oligo dT columns (Micro-FastTrack; Invitrogen,
San Diego, CA). cDNA was then synthesized from the mRNA
of 20,000 AM, 100,000 PBMC, or 100,000 IPMC using Maloney
murine leukemia virus reverse transcriptase and random hexamer
oligonucleotides (2, 17). After a 2-rain, 94~ denaturation step,
between 34 and 38 cycles of PCR were conducted (45 s, 94~
denaturation; 45 s, 60~ annealing; and 2 min, 72~ extension)
on cDNA from 1000 AM, 10,000 IPMC, or 10,000 PBMC. All
cDNA samples were mixed with aliquots of the same PCR master
mix using appropriate cytokine MIMICs (Clontech, Palo Alto, CA)
as internal controls for standardization of PCR product (2, 18).
Cytokine primers (Clontech) were used at 0.4 #M. To detect
Published February 1, 1995
0.08
a
0.06
0
200
PE
0.04
.PA
0.02
Neutrophil Phosphatidic Acid
250
~Pl
I1~
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PC
~ SM
yntreated
150
100
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0.00
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0
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b
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-50
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hemorrhage, mice resuscitated with CT1501R had fewer
(p <0.05) pulmonary interstitial neutrophils than untreated
mice (Fig. 4 c) and the percentage of BAL leukocytes that
were neutrophils in lungs of CT1501R treated mice (6 -+
2%) was less (v <0.05) than the percentage of neutrophils
in untreated mice (14 _+ 2%). The decreased injury and
inflammation in lungs of CT1501R treated mice was corroborated by histological analysis (Fig. 5).
For unknown reasons, increased lung cytokine levels are
a prominent feature in patients with ARDS and in mice subjected to hemorrhage-resuscitation (2, 4, 20, 21). We found
that alveolar macrophages (Fig. 6 a), blood monocytes (Fig.
6 b) and intraparenchymal lung mononuclear cells (Fig. 6 c)
from hemorrhaged mice resuscitated with CT1501R had decreased (p <0.05) mRNA levels for TNF-ot compared to untreated, hemorrhaged and resuscitated mice. Similarly, inAbraham et al.
0
[HypoxiaI
t
Figure 1. Effect of hypoxia reoxygenation on neutrophil PA metabolism. Representative experimental HPLC tracings for untreated, control
neutrophils (a), untreated neutrophils exposed to hypoxic conditions for
60 min and then reoxygenation for 20 min (b), and neutrophils incubated
with 10 #M CT1501R, then exposed to hypoxia-reoxygenation (c). Control neutrophils synthesized little PA or lyso-PA, whereas untreated, hypoxia-reoxygenated neutrophils synthesized a variety of PA species, particularly Rs
min PA. In contrast to PA, neutrophils treated with CT1501R
accumulated lyso-PA species, with attenuation in synthesis of PA species,
particularly Rf 4-8 min. Consistent with published results (9), phosphatidyl
inositol (PI) mass was diminished by hypoxia-reoxygenation (b), and this
process was not inhibited by adding CT1501R (c).
571
-60
.
CT1501R
2"o
Reoxygenation
25
3o
3"5
]
Minutes
treatment
Figure 2. Effectof hypoxia-reoxygenation on neutrophil PA metabolism. CT1501R treatment inhibited PA increases and lyso-PA decreases
in human neutrophils subjected to hypoxia-reoxygenation in vitro. (a) After
hypoxia-reoxygenation, untreated neutrophils had a rapid rise in Rf 4-8
min PA (enriched in sn-2 linoleoyl moieties and sn-1 acyl C18 saturated
and unsaturated moieties) and essentially a partially sustained or oscillatory production of PA. Adding 10 or 100/~M CT1501R to neutrophils
at the onset of hypoxia blunted PA production. (b) Hypoxia-reoxygenated,
untreated neutrophils had little Rf 2-3 min lyso-PA (enriched in sn-1 acyl
C18 saturated and unsaturated moieties), whereas CT1501R treated neutrophils accumulated lyso-PA. Lipid masses are determined by absorption
at 217-222 nm, and then normalized to total mass. The masses shown
are averaged from five experiments where each time point was performed
in triplicate, with error bars showing SEM.
traparenchymal lung mononudear cells from hemorrhaged
mice resuscitated with CT1501R had decreased ~ <0.05)
amounts of mRNA for IL-13 (Fig. 6 d), IL-6 (Fig. 6 e), and
IFN-7 (Fig. 6 f ) compared to untreated, hemorrhaged and
resuscitated controls. In paraUel, BAL recovered from hemorrhaged and CT1501R resuscitated mice had less ~ <0.05)
TNF-o~ (Fig. 6 g) and IFN-7 (Fig. 6 h) activity than BAL
from untreated, hemorrhaged, and resuscitated mice. Thus,
resuscitation with CT1501R after hemorrhage decreased the
expression and activity of a number of different cytokines
in circulating mononuclear cells and resident lung cells.
Our results show that PA may fundamentally mediate
multiple events responsible for the development of acute lung
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~." 0.0~
0
0,02
CT-1501R
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0.0(
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Neutrophil Lyso - phosphatidic Acid
0.02
Published February 1, 1995
Neutrophil Adherence
80
2.0
60
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0'3
0.5
20
Interstitial Lung Edema
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Intraalveolar Hemorrhage
2.5
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Neutrophil Superoxide Production
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Hem
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CTIS01R
Figure 4. Effect of LPAAT inhibition of lung injury in mice subjected
0
m
Unstimulated
neutrophils
r
Stimulated
neutrophils
Stimulated
neutrophils
+
CT1501R
Figure 3. Effect of LPAAT inhibition of neutrophil function in vitro.
Adding CT1501R (7/zM) decreased (p <0.05) stimulated human neutrophil adherence (a) and chemotaxis (b), but not superoxide anion generation
(c), in vitro. Each value is the mean _+ SE of six or more determinations
compared by analysis of variance and corrected by Student-Newman-Keuls
test for differences between groups using a/~ value of <0.05 as significant.
injury after hemorrhage and resuscitation. CT1501R treatment after blood loss decreased lung injury and lung neutrophil accumulation in mice subjected to hemorrhage-resuscitat i o n - a finding paralleled by CT1501R treatment decreasing
neutrophil function in vitro. Our results also suggest that
systemic hypoxia followed by tissue reoxygenation, such as
occurs in hemorrhage-resuscitation, induces the formation
of PA in neutrophils as well as in lung tissue. Increased pro-
572
to hemorrhage and resuscitation. Hemorrhaged mice treated with CT1501R during resuscitation had reduced (p <0.05) interstitial lung edema
(a), intraalveolar hemorrhage (b), and neutrophil accumulation (c), compared to untreated mice subjected to hemorrhage-resuscitation. Treatment
with CT 1501R (tOO mg/kg given in 0.2 ml PBS) or 0.2 ml PBS i.v. was
started 1 h after hemorrhage or cardiac puncture, and then was given i.p.
every 8 h for a total of 9 doses over 72 h. Each mean +_ SE for 6-8 mice
was analyzed by one way analysis of variance with the Tukey-Kramer multiple comparison test using a p value of <0.05 as significant.
duction of PA then induces neutrophil chemotaxis, proinflammatory cytokine generation and lung injury. Although the
present experiments revealed that inhibition of LPAAT with
CT1501R decreased in vitro neutrophil chemotaxis and adherence induced by zymosan and PMA, additional studies
will be necessary to determine whether inhibition of neutrophil functions occurs when other signaling pathways are activated, particularly in the in vivo setting.
CT1501R also abrogated the induction of mRNA for several proinflammatory cytokines in multiple cell types in the
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0
Published February 1, 1995
Hemorrhage
Control
Hemorrhage
+ CT-1501R
lungs of mice subjected to hemorrhage-resuscitation. The
protean effects and extensive protection which occurred after
CT1501K treatment is most likely explained by the basic signaling nature of PA on diverse components of the inflammatory cascade. Inflammatory stimuli translocate LPAAT to the
plasma membrane (7); therefore, it is possible that translocation and activation of LPAAT also occurs after oxidative stress.
PA produced by LPAAT may also directly activate atypical
protein kinase C species or alternative kinases and may alter
GTPase activating protein/ras interactions (22-24). Adding
PA to cells also produces potent mitogenic effects, stimulates
calcium flux and phospholipase C activity, and induces expression of several protooncogenes and growth factors (25-27).
At our dose of CT1501R, inhibition of LPAAT appears to
be the primary intracellular signaling pathway responsible
for the observed effects on neutrophil function and lung injury, although it is possible that diacylglycerols formed from
PA by phosphatidate phosphohydrolase rather than PA itself
are the relevant signaling molecules affected by CT1501R.
The ICs0 for inhibiting phosphodiesterase by CT1501R is
between 100 and 500 #M in vitro systems, significantly above
concentrations (i.e., 10-30 #M) at which CT1501K effectively inhibits PA synthesis (6, 8). It is notable that PI hy-
573
Abrahamet al.
drolysis and DG/IP3 formation, as well as Ca +z flux, are
not inhibited by CT1501R (8). Similarly, sphingomyelin hydrolysis to ceramide and TNF-ot induced apoptosis via NF-kB
are not inhibited by CT1501R (28).
Lyso-PA is a potent mitogenic stimulus through ras/MAPK
and rho activation via an external receptor which functions
to activate G proteins (29). However, intracellular injection
of lyso-PA appears to lack the activating effects induced by
interactions of lyso-PA with its external receptor (7, 30). Therefore, it is unlikely that intracellular accumulation of lyso-PA
through LPAAT inhibition by CT1501K plays an important
counter-regulatory role in intracellular signaling.
Our findings suggest that inhibiting PA may have potential in the treatment and prevention of conditions, such as
ARDS and septic shock, which are associated with activation of a proinflammatory cytokine cascade (2, 8, 10, 20, 21,
31). Because PA proximally mediates a basic intracellular signaling mechanism, inhibiting PA with CT1501R may be more
effective therapeutically than agents which have more discrete effects. For example, the interleukin-1 receptor antagonist
decreases only II,-1 effects and has no direct effect on neutrophil function in vitro (32).
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Figure 5. Effectof LPAATinhibition on lung histologyin mice subjectedto hemorrhage and resuscitation. Lungs from hemorrhaged mice treated
with CT1501Rduring resuscitationhad reducedhistologicabnormalitiescomparedto lungs from untreated mice subjectedto hemorrhage-resuscitation.
Representative hematoxylin and eosin stained pulmonary sectionsobtained 3 d after cardiac puncture in mice subjectedto methoxyfluraneanesthesia
and cardiacpuncture, without blood withdrawal, followed1 h later by retroorbital injection of 5 U heparin in 0.1 ml PBS (Control),in mice subjected
to 30% blood volume hemorrhage and then resuscitated with the shed blood 1 h after (Hemorrhage)and in mice subjected to 30% blood volume
hemorrhage and then resuscitated with the shed blood 1 h later, but treated with CT1501R.every 8 h starting 1 h after hemorrhage (Hemorrhage
+ CT1501K). The pulmonary histology in control mice is not different from that seen in normal, unmanipulated and unhemorrhaged mice.
Published February 1, 1995
Alveolar Macrophage T N F - a
m R N A Levels
2.0
r
1.5
a~
i
1.0
I1
<
0.5
?,
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lntraparenchymal Lung Mononuclear Cell IL-6
m R N A Levels
4
O
~
<
g
T
~
e
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1
0
Blood Mononuclear Cell T N F - a
.~
0.4
=.
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b
m R N A Levels
Intraparenchymal Lung Mononudear Cell I F N - y
m R N A Levels
f
.'~ 2.0
0.3
~
0.2
g
=
1.5
o.1
<
o
~
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~
o
lntraparenchymal Lung Mononudear Cell T N F - a
5 c
m R N A Levels
Lung Lavage TNF-a Activity
4000
T
r
~ 2000
21 L
lOOO
"~
0
lntraparenchymal Lung Mononuclear Cell I L - I ~
m R N A Levels
3
d
200
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150
e~
..D
<
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Lung Lavage 1 F N - ' y Activity
0
I
T
I
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T IIT
Control
+
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Hem
§
CT1501R
100
50
l
0
Conizol
Conb'ol
+
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T
Hem
Hem
+
CT1501R
Figure 6. Effectof LPAATinhibition on lung cytokinesin mice
subjected to hemorrhage and
resuscitation. Hemorrhaged mice
which were resuscitated with
CT1501R had decreased (p <0.05)
amounts of mRNA for TNF-~ in
their alveolarmacrophages (a) and
blood mononuclear cells (b), decreased mRNA levels for TNF-a
(c), IL-1/~(d), Ir-6 (e), and IFN-'y
(f) in their intraparenchymallung
mononuclear cells, and decreased
TNF-ct (g) and IFN-'y (h) activity
in their bronchoalveolar lavage
(BAL) compared to untreated hemorrhaged-resuscitated mice. The
mean _+ SE for 6-8 mice was analyzed by one way analysis of variance with the Tukey-Kramermultiple comparison test using p
(0.05 as significant.
This work was funded in part by the Swan Foundation, American Heart Association, and the National
Institutes of Health (RO1-HL45582, P50 HL40784, GM39102) and a gift from Marc Arnold and Barbara
Pfifferling.
Address correspondence to Edward Abraham, M.D., Box C272, 4200 East Ninth Ave., Denver, CO 80262.
Receivedfor publication 2June 1994 and in revisedform 22 August 1994.
574
Lung Inflammatory Mechanisms in Shock
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Published February 1, 1995
575
Abrahamet al.
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